1. Field of the Invention
The present invention relates to novel methods for identifying fungal pathogens in a biological sample. In particular, this invention relates to methods for screening biological samples for the presence of fungal pathogens using hybridization methods and probes capable of sensitively and specifically detecting and distinguishing nucleic acid sequences unique to fungi. Also provided are antibodies capable of binding selectively to fungal proteins.
2. Background of the Invention
Candida albicans (hereinafter referred to as xe2x80x9cC. albicansxe2x80x9d), once considered a relatively minor fungal pathogen, has recently become a particularly serious health concern as the causative agent of candidosis (also called candidiasis). The incidence of C. albicans infections is rising rapidly with the increase in immune deficiency diseases and immunosuppressive therapy (Bodey and Fainstein, In Systemic Candidiasis, pp. 135 (Eds., Raven Press, New York 1985). Candidosis is a common nosocomial infection afflicting both immunosuppressed and postoperative patients. (Holmes, A. R., et al. Yeast-specific DNA probes and their application for the detection of Candida albicans, J. Med. Microbiol., 37:346-351 (1992)). Although candidosis is a particular concern among immunocompromised individuals, Candida infections are not limited to this group. C. albicans is the major opportunistic fungal pathogen in humans (odds, F. C., In Candida and candidosis. (Ed.) Leicester University Press, Leicester, United Kingdom (1989)) and is capable of establishing infection whenever the host immune system or normal flora are perturbed.
Although the C. albicans species is a particular health concern, other species of the Candida genus are also pathogenic. The genus Candida is comprised of approximately 200 diverse yeast species classified together due to their lack of a sexual cycle (Meyer et al., In Genus 4, Candida, pp. 1-12, (Ed.) N. J. W. Kregervan Riij, Elsevier, Amsterdam (1984)). A minority of Candida species are pathogenic and 80% of the clinical isolates are either C. albicans or C. tropicalis (Hopfer, R. L. In Mycology of Candida Infections, G. P. Bodey. an V. Fainstein (eds.), Raven Press, New York (1985)).
In immunocompromised hosts, candidosis is a life threatening condition. The prognosis for a patient infected with C. albicans can be improved markedly, however, with prompt antifungal treatment. Treatment may be delayed until a positive diagnosis of Candidosis is obtained since antifungal drugs are toxic. (See Holmes, et al., 1992.)
Diagnostic tests for the identification of C. albicans or other fungal pathogens in vivo often require complete cultural identification protocols (Musial et al., Fungal Infections of the Immunocompromised Host: Clinical and Laboratory Aspects, Clin. Microbiol. Rev. 1:349-364 (1988)). Methods currently used for the diagnosis of fungal pathogens include: cultural identification, biopsy, serodiagnosis, identification of metabolites, isoenzyme determination, pulsed field gel electrophoresis and analysis of restriction fragment length polymorphisms. Most of these methods are time consuming, laborious and provide inconclusive results.
Potential methods for diagnosing fungal infections through DNA screening have focused on detecting specific nucleotide sequences such as ribosomal DNA (Hopfer, R. L. et al., Detection and differentiation of fungi in clinical specimens using polymerase chain reaction (PCR) amplification and restriction enzyme analysis, J Med. Vet. Pharm. 31:65-75 (1993)) and the p450 genes (Buchman, T. G. et al., Detection of surgical pathogens by in vitro DNA amplification. Part I, Rapid identification of Candida albicans by in vitro amplification of a fungal specific gene. Surgery, 108:338347 (1990)). However, no commercial diagnostic techniques embodying methods related to the identification of these genes in biological samples are known.
The sequences of approximately 1800 C. albicans genes are available in computerized databases. The relatively small amount of fungal specific or unique genetic information available for C. albicans places limitations upon the number of DNA sequences that can be used as targets for screening probes and concomitantly reduces the likelihood of identifying a sequence unique to fungi and amenable to identification through DNA screening techniques. For example, very few of available sequences are from genes involved in fungal amino acid biosynthesis pathways. One impediment to developing nucleic acid based screening techniques for Candidosis is that basic information about uniquely fungal metabolic pathways and cognate genes of C. albicans is lacking (Kurtz et al., Molecular Genetics of Candida Albicans, pp. 21-73, Kirsch, Kelly and Kurtz (eds.) CRC Press Inc. Boca Raton, Fla. (1990)).
Similar impediments exist to developing immunological methods of identifying a fungus present in a biological sample. Relatively few antigenic determinants unique to fungi are known, and none are believed to have been successfully utilized as targets for antibody binding in commercially available form.
Among the proteins that have been studied in C. albicans and other pathogenic fungi are the enzymes that make up the xcex1-aminoadipate pathway for the biosynthesis of lysine. This unique pathway has only been identified in Phycomycetes, Euglenids, yeasts and other higher fungi (Bhattacharjee, The xcex1-aminoadipate Pathway for the Biosynthesis of Lysine in Lower Eukaryotes, CRC Critical Rev. in Microbiol. 12:131-151 (1985); Lejohn, Enzyme Regulation. Lysine Pathways and Cell Wall Structures as Indicators of Evolution in Fungi, Nature 231:164-168 (1971); and Vogel, Two Modes of Lysine Synthesis Among Lower Fungi: Evolutionary Significance, Biochim. Biophys. Acta 41:172-174 (1960); (Garrad, R. Masters Thesis, Miami University (1989) and, Garrad and Bhattacharjee, Lysine biosynthesis in selected pathogenic fungi: Characterization of lysine auxotrophs and the cloned LYS1 gene of Candida albicans, J. Bacteriol. 174:7379-7384 (1992)). Lysine biosynthesis is an example of a biochemical divergence between higher fungi, which use the xcex1-aminoadipic acid pathway (distinct from the diaminopimelic acid pathway used by bacteria and plants), and human host cells, which cannot synthesize lysine. The aminoadipate pathway for lysine biosynthesis, therefore, offers a unique opportunity to develop molecular probes for detection of fungal pathogens and as a potential drug target.
The xcex1-aminoadipate pathway consists of eight enzyme catalyzed steps; there appear to be seven free intermediates in S. cerevisiae (Bhattacharjee, The xcex1-aminoadipate pathway for the biosynthesis of lysine in lower eukaryotes, CRC Critical Review in Microbiol. 12:131-151 (1985)). An understanding of the genetics, biochemical aspects, and regulation of the xcex1-aminoadipic acid pathway has been obtained by studies in the model organisms Saccharomyces cerevisiae, Schizosaccizaromyces pombe, and in the yeast Candida maltosa (Bhattacharjee 1992; Feller et al. 1994; Hinnebusch 1992; Schmidt et al. 1985). The final reversible step of the xcex1-aminoadipate pathway is catalyzed by saccharopine dehydrogenase, which is encoded by the LYS1 gene of S. cerevisiae and C. albicans, and the LYS5 gene of Y. Lipolytica (Fujioka, Chemical mechanism of saccharopine dehydrogenase (NAD, L-lysine forming) as deduced from initial rate pH studies, Arch. Biochem. Biophys. 230:553-559 (1984); Garrad and Bhattacharjee, Lysine biosynthesis in selected pathogenic fungi: Characterization of lysine auxotrophs and the cloned LYS1 gene of Candida albicans, J. Bacteriol. 174:7379-7384 (1992); and Xuan et al., Overlapping reading frames at the LYS5 locus in the yeast Yarrowia lipolytica, Mol. Cell. Biol. 10:47954806 (1990)).
The conversion of aminoadipic acid to [xcex1-aminoapidate] xcex1-aminoadipate semialdehyde is an obligatory step for the biosynthesis of lysine in yeast and is catalyzed by the enzyme, xcex1-aminoadipate semialdehyde dehydrogenase, commonly known as xcex1-aminoadipate reductase (AAR) (Bhattacharjee 1985; Broquist 1971). AAR is a heterodimeric enzyme encoded by two unlinked genes LYS2 and LYS5 in S. cerevisiae, the equivalent genes in S. pombe being lys1+ and lys7+(Rajnarayan et al. 1992; Sinha and Bhattacharjee 1970; Ye and Bhattacharjee 1988).
The necessity of methods that provide rapid, sensitive and selective detection of fungal pathogens in biological samples and particularly for detection of C. albicans in biological samples increases each year is understood by those skilled in the art. The increasing use of immunosuppressive drugs in connection with organ transplants, autoimmune diseases, A cancer, and the increasing number of patients suffering from acquired immunodeficiency syndrome, have resulted in a dramatic increase in the incidence of candidosis and other fungal infections. Because fungal infections are life threatening, physicians may prescribe antifungal drugs even in the absence of a definitive diagnosis. Due to the sometimes toxic effects of such drugs, however, their administration without such a definitive diagnosis is undesirable. Provided herein is a methodology for identification of a fungal pathogen in a biological sample by detection of nucleic acid sequences unique to fungi. The present invention fills a need in the art such that rapid identification of a fungal pathogen may be accomplished. As such, treatment of affected patients may be begun more rapidly that the art currently allows. Using the methods of the present invention, a more favorable prognosis may be associated with fungal diseases.
An object of this invention is to provide method of identifying the presence of a pathological fungus in a biological sample. It is an additional object of the invention to provide nucleic acid constructs for use in screening biological samples for the presence of fungal pathogens.
It also an object of the invention is to provide such nucleic acid constructs comprising nucleotide sequences that are specific to fungal organisms, preferably those causing pathological consequences in a host. Yet another object of the present invention is to provide improved materials and reagents for use in screening biological samples for the presence of C. albicans. It is a further object of the present invention to provide nucleic acid constructs for use in screening biological samples for the presence of C. albicans. 
Another object of the invention is to provide antibodies for use in screening biological samples for the presence of fungal pathogens. It is another object of the invention is to provide antibodies that are sensitive and specific for fungal proteins, preferably proteins of fungi having pathological effects in a host. It is a further object of the present invention to provide antibodies for use in screening biological samples for the presence of C. albicans. 
In one embodiment, the present invention provides novel nucleic acids, reagents and PCR primers capable of selectively amplifying a nucleotide sequence found in fungal genomic DNA but not in higher eukaryote genomic mateial. In a preferred embodiment, the nucleic acids and PCR primers are derived from genomic DNA of C. albicans. In a more preferred embodiment, the present invention provides PCR primers and methodologies for sensitively and selectively amplifying LYS2 (US 8133; SEQ ID NO.: 1) nucleotide sequence from a biological sample containing C. albicans DNA.
In one preferred embodiment, nucleic acid hybridization probes are provided which comprising a nucleotide sequence illustrated in SEQ ID NO.:7 and homologues or labeled variants thereof.
In another preferred embodiment, the nucleic acid sequence comprises the sequence:
VB21 5xe2x80x2-TTAACAAAGAGATTGTTT-3xe2x80x2 (SEQ ID NO.:2)
VB22 5xe2x80x2-CTGAAACCTCTAATCTT-3xe2x80x2 (SEQ ID NO.: 3) and homologues or labeled variants thereof.
In yet another embodiment, peptide sequences are provided from which antibodies may be generated to detect fungus-specific polypeptides in a biological sample. In a preferred embodiment, the peptide sequences are derived from xcex1-aminoadipate reductase. In a more preferred embodiment, the peptide sequence comprises LTKRDCLKIRGFT (SEQ ID NO.: 4).
In another embodiment, the invention provides methods of using antibodies reactive to a fungus-specific peptide in a biological sample. In a preferred embodiment, a method of detecting fungal xcex1-aminoadipate reductase encoded by LYS2 is provided. The invention additionally provides novel antibodies that may be labeled for use in a detection assay such as the enzyme-linked immunosorbent assay (ELISA). In an alternative embodiment, these epitopes may be labeled and used to detect the presence of a fungus in a biological sample, for example, by competitively inhibiting antibody binding in a radioimmunoassay. Reagents and kits comprising the inventive antibodies and epitopes are also provided.